The long term objective of this project is to identify the transmitter or transmitters at synapses in mammalian taste buds. Taste receptor cells make synaptic connections with afferent terminals from sensory ganglion cells. Taste receptor cells also are believed to form lateral synaptic connections with other taste bud cells in the peripheral organs of taste, i.e., intrinsic or intragemmal synaptic interactions. Additionally, efferent axons may synapse with taste bud cells. By analogy with other sensory organs, one might speculate that glutamate (glu), acetylcholine (Ach), serotonin (5HT) and norepinephrine (NE) are released at these synaptic sites. However, the identity of neurotransmitters in taste buds is not yet known. This lack of information has greatly slowed an analysis of peripheral taste mechanisms in health and disease. We propose 3 Specific Aim to fill this gap in information about taste bud synapses. Fist, we will use quantitative autoradiography and immunocytochemistry to study the uptake of 5HT, NE and glu in mouse taste buds. We will test if there are specific re-uptake mechanisms in taste cells for these transmitter candidates. In collaboration with J. Kinnamon (Project #3), we will determine the ultrastructure of the taste cells that accumulate these transmitters and correlate transmitter identification with synaptic markers. Second, we will use the same techniques along with new biosensor methodologies and carbon fiber electrode voltammetry, to test whether 5HT, NE, glu and ACh are released by synapses in mouse taste buds. We will determine whether the release is Ca-dependent, and whether stimulating taste buds with taste stimuli (sweet, bitter, salty, etc.) releases different transmitters. One series in these experiments will be conducted in the laboratory of S. Kinnamon (Project #5). Third, we will employ immunocytochemistry and in situ hybridization to examine the expression of transmitter receptors in taste buds and their innervating sensory neurons. Although the focus of our Specific Aims is on glu, ACh, 5HT and NE, we will keep an open mind and explore the possibility that transmitters other than these may be released. At the end of the project, we will have identified one or more of the key neurotransmitters released at synaptic sites in mammalian taste buds.